Conversion of Glucosinolates to Isothiocyanates in Humans After Ingestion of Cooked Watercress1
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Vol. 8, 447–451, May 1999 Cancer Epidemiology, Biomarkers & Prevention 447 Conversion of Glucosinolates to Isothiocyanates in Humans after Ingestion of Cooked Watercress1 Serkadis M. Getahun and Fung-Lung Chung2 vegetables. Furthermore, upon incubation of the cooked Division of Carcinogenesis and Molecular Epidemiology, American Health watercress juice with fresh human feces under anaerobic Foundation, Valhalla, New York 10595 conditions, ;18% of glucosinolates was hydrolyzed to ITCs in 2 h. These results suggest that the microflora in the intestinal tract are a likely source for the hydrolysis Abstract of glucosinolates to ITCs in humans. Isothiocyanates (ITCs), major constituents of cruciferous vegetables, can inhibit tumorigenesis in rodents by Introduction modulating the metabolism of carcinogens. ITCs that ITCs,3 which occur as major constituents in cruciferous vege- occur as glucosinolates are released by myrosinase- tables, have been shown to inhibit tumorigenesis in rodents mediated hydrolysis when raw vegetables are chopped or treated with carcinogens (1–4). Studies in vitro and in vivo chewed. However, because cruciferous vegetables are showed that the inhibitory activity of ITCs against carcinogen- commonly consumed by humans after being cooked, it is esis can be attributed to their ability to deactivate phase I important to examine whether dietary glucosinolates are enzymes and/or to activate phase II enzymes (3, 5). Recent converted to ITCs after cooked cruciferous vegetables in studies in humans also demonstrated that the consumption of which myrosinase is deactivated have been consumed. cruciferous vegetables resulted in an increase in glutathione This information is useful for evaluating the potential transferase activity and a decrease in the metabolic activation of role of ITCs in cruciferous vegetables in the protection carcinogens in smokers (6–8). These results support a potential against human cancers. A urinary marker, based on a role of ITCs in cruciferous vegetables in the prevention of cyclocondensation product formed by the reaction of human cancers. ITCs and their conjugates with 1,2-benzenedithiol, was ITCs are products of degradation of glucosinolate precur- used to quantify the uptake of dietary ITCs in humans. sors in cruciferous vegetables, a reaction usually catalyzed by At breakfast and lunch, nine volunteers consumed a total myrosinase, an enzyme that is activated upon crushing the of 350 g of cooked watercress in which the myrosinase vegetables by chopping or chewing (Fig. 1a; Refs. 9 and 10). activity was completely deactivated. On the basis of the We and others have demonstrated that significant amounts of analysis of ITCs in the cooked watercress upon adding ITCs are released after ingesting raw cruciferous vegetables, as exogenous myrosinase, the amount of glucosinolates m indicated by the presence of ITC mercapturic acid in the urine ingested by each subject was estimated to be 475 mol. (11–13). Little is known, however, about the fate of glucosi- The 24-h urine samples showed that the total urinary nolates in humans upon consumption of cooked vegetables in excretion of ITC conjugates in the subjects ranged from m which the myrosinase activity is absent. Because crucifers are 5.6 to 34.8 mol, corresponding to 1.2–7.3% of the total commonly consumed by humans after being cooked, it is im- amount ingested. On the basis of our previous results ; portant to know whether glucosinolates in cooked vegetables that 50% of dietary ITCs were excreted in the urine as are converted to ITCs. This information can be useful in the conjugates, these values represent the minimal in vivo investigation of the potential roles of dietary ITCs in human conversion of glucosinolates to ITCs. For purposes of cancers. Here, we studied the metabolic conversion of glucosi- comparison, we carried out a second experiment in which nolates to ITCs in humans after ingesting cooked and uncooked 150 g of uncooked watercress were consumed. The watercress and compared the extent of conversion. Because percentage of urinary ITC conjugates excreted in this ITCs are primarily metabolized via the mercapturic acid path- study ranged from 17.2 to 77.7% of the total ingested way, the N-acetylcysteine conjugate of ITC in the urine serves ITCs. These results indicate that glucosinolates are as a marker of ITCs released from glucosinolates. Using a converted to ITCs in humans after ingestion of cooked previously developed assay based on the reaction of ITCs or watercress, in which the myrosinase has been completely their thiol conjugates with 1,2-benzenedithiol forming a cyclic inactivated. The extent of conversion, however, is dithiol thione product (Fig. 1b; Refs. 14 and 15), we quantified considerably less than that after ingesting uncooked the urinary metabolites of ITCs after eating watercress with and without myrosinase activity. In addition, we also examined whether intestinal microflora are a possible source for the Received 10/7/98; revised 2/22/99; accepted 3/8/99. degradation of glucosinolates in humans by incubating cooked The costs of publication of this article were defrayed in part by the payment of watercress juice with human fecal samples. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by NCI Grant CA46535. This is Paper No. 30 in the series “Dietary Inhibitors of Chemical Carcinogenesis.” 2 To whom requests for reprints should be addressed, at American Health Foun- dation, 1 Dana Road, Valhalla, NY 10595. Phone: (914) 789-7161; Fax: 3 The abbreviations used are: ITC, isothiocyanate; HPLC, high-performance (914) 592-6317; E-mail: [email protected]. liquid chromatography. Downloaded from cebp.aacrjournals.org on September 25, 2021. © 1999 American Association for Cancer Research. 448 Watercress and Dietary ITCs Table 1 Cumulative amounts of ITC conjugates in 24-h human urine before (baseline) and after ingestion of cooked watercress meal Urine excreted before ITCs in baseline urine Urine excreted after ITCs in urine after % of administered dose excreted Subject no. Sex consumption (liters) (mmol)a consumption (liters) consumption (mmol)a as ITC conjugatesb 1 M 1.2 5.4 6 0.2 2.0 34.8 6 0.8c 7.3 2 F 0.6 0.6 6 0.2 1.5 33.0 6 2.5 6.9 3 F 1.5 0.5 6 0.1 3.2 28.1 6 0.2 5.9 4 F 1.3 1.1 6 0.1 0.8 17.8 6 0.7 3.7 5 M 2.8 1.0 6 0.1 3.0 17.4 6 0.1 3.6 6 F 0.3 0.3 6 0.1 0.7 15.2 6 0.6 3.2 7 F 1.4 1.6 6 0.4 1.4 15.4 6 0.3 3.2 8 M 2.1 2.2 6 0.1 1.4 5.6 6 0.1 1.2 9 M 1.8 0.7 6 0.1 3.0 5.7 6 0.2 1.2 a Mean 6 SD (triplicate data). b On the basis of 475 mmol of ITCs in cooked watercress juice treated with myrosinase. c Values obtained after subtracting the baseline concentration. Table 2 Experimental Protocol Days 1 and 2 Day 3 Day 4 Baseline Watercress consumption Follow-up urine sample was collected on day 2 as baseline prior to the consumption of watercress on day 3. Fresh watercress (Nas- turtium officinale) purchased from a local supermarket was cooked in boiling water for 3 min (85.7 g/liter), and a total of 350 g was ingested by each volunteer, 200 g at breakfast and 150 g at lunch. After breakfast, a 27-h urine sample was collected from each subject. Another 18-h urine sample was collected after lunch on day 4 as the follow-up. After a 7-day period, a second experiment was conducted. The same protocol was used with the exception that uncooked watercress was consumed. Because we have previously conducted a similar study using uncooked watercress (12), in this experiment, only four subjects (subjects 1, 2, 7, and 8; see Table 3) participated who consumed 100 g for breakfast and 50 g for lunch on day 3. In both experiments, a 10-ml aliquot of each pooled urine sample was stored at 220°C until ITC analysis. Analysis of ITC in Urine. The amount of ITCs in the urine Fig. 1. a, myrosinase-catalyzed conversion of glucosinolates to ITCs via Lossen samples was analyzed by a previously described method (15). rearrangement. b, reaction of ITC with 1,2-benzenedithiol forming the cyclocon- densation product, 1,3-benzodithiole-2-thione, and amine. Briefly, the urine sample was thawed and vortexed. A 1-ml aliquot was placed in a 2-ml centrifuge tube and centrifuged for 20 min at 1000 3 g. An aliquot (100 ml) of the supernatant was mixed with 600 ml of 1,2-benzenedithiol in 2-propanol (10 mM, Materials and Methods degassed) and 500 ml of phosphate buffer (0.1 M, pH 8.5, Chemicals. 1,2-Benzenedithiol was purchased from Lancaster degassed) in a 2-ml Chromacol autosampler vial with a screw Synthesis, Inc. (Windham, NH). 1,3-Benzodithiole-2-thione cap (Chromacol, Inc., Trumbull, CT). The mixture was then was prepared and characterized according to a published vortexed for 1 min and incubated at 65°C for2hinawater bath method (14, 15). Myrosinase was prepared from mustard seeds shaker. The product 1,3-benezodithiol-2-thione was analyzed m 3 according to a published procedure (16). Nutrient broth me- by reverse-phase HPLC using a Waters Bondapak C18 (150 m dium was purchased from Difco Laboratories (Detroit, MI). 3.9 mm) with a C18 Waters Bondapak guard column and a Sodium thioglycolate was purchased from Fisher Scientific detection wavelength of 365 nm.